Interactions between postranslational protein modifications were studied in rat brain cytosol and synaptic membranes. Protein-O-Carboxylmethyltransferase\(PCM; E.C. 2.1.1.24) using S-adenosylmethionine (AdoMet) as a methyl donor, transfers methyl groups to aspartic and glutamic acid residues on proteins. Ca++-calmodulin-dependent protein kinases transfer phosphate groups to proteins, using ATP as a phosphate donor. Preincubation of calmodulin with PCM resulted in the formation of methylesters on calmodulin, carboxylmethylated calmodulin was less able to stimulate Ca++-calmodulin protein kinase in cytosol and synaptic membranes. When the protein kinase fraction was carboxylmethylated, and native calmodulin used to activate Ca++-stimulated phosphorylation, inhibition of phosphorylation was also noted. These results suggest that protein carboxylmethylation can modulate the activity of Ca++-calmodulin-dependent protein kinase.